REVIEWS

Functions and dysfunctions of

mitochondrial dynamics Scott A. Detmer and David C. Chan Abstract | Recent findings have sparked renewed appreciation for the remarkably dynamic nature of mitochondria. These organelles constantly fuse and divide, and are actively transported to specific subcellular locations. These dynamic processes are essential for mammalian development, and defects lead to neurodegenerative disease. But what are the molecular mechanisms that control mitochondrial dynamics, and why are they important for mitochondrial function? We review these issues and explore how defects in mitochondrial dynamics might cause neuronal disease.

Cristae In the 1950s, seminal electron microscopy studies led shape of mitochondria, fusion and fission are crucialInvaginations of the to the canonical view of mitochondria as bean-shaped for maintaining the functional properties of the mito-mitochondrial inner organelles. These studies revealed the ultrastructural chondrial population, including its respiratory capacity.membrane. hallmarks of mitochondria, which include double lipid Moreover, mitochondrial dynamics has key roles inNebenkern structure membranes and unusual inner membrane folds termed mammalian development, several neurodegenerativeA cytosolic structure, found in cristae . Recent studies have led to renewed appre- diseases and apoptosis.some insect spermatids, that is ciation for the fact that the mitochondrial structureformed by the fusion of is highly dynamic1,2. Mitochondria have drastically Mitochondria as dynamic organellesmitochondria. different morphologies depending on the cell type By several criteria, mitochondria are dynamic and, even in the same cell, mitochondria can take organelles. First, the shape and size of mitochondria on a range of morphologies, from small spheres or are highly variable and are controlled by fusion and short rods to long tubules. In fibroblasts, for example, fission. Second, mitochondria are actively transported mitochondria visualized with fluorescent proteins or in cells and they can have defined subcellular distribu- specific dyes typically form tubules with diameters of tions. Finally, the internal structure of mitochondria ~0.5 mm, but their lengths can range from 1–10 mm can change in response to their physiological state. or more. Even more remarkably, imaging studies in live Dynamic shape. The length, shape, size and number cells indicate that mitochondria constantly move and of mitochondria are controlled by fusion and fission undergo structural transitions. Mitochondrial tubules (FIG. 1a). At steady state, the frequencies of fusion and move with their long axes aligned along cytoskeletal fission events are balanced 4 to maintain the overall tracks3. Individual mitochondria can encounter each morphology of the mitochondrial population. When other during these movements and undergo fusion, this balance is experimentally perturbed, dramatic resulting in the merging of the double membranes and transitions in mitochondrial shape can occur. Genetic the mixing of both lipid membranes and intramito- studies in yeast and mammals indicate that cells with chondrial content (BOX 1). Conversely, an individual a high fusion-to-fission ratio have few mitochondria, mitochondrion can divide by fission to yield two or and that these mitochondria are long and highly inter- more shorter mitochondria. connected5–8 (FIG. 2). Conversely, cells with a low fusion- What are the molecular mechanisms that underlie to-fission ratio have numerous mitochondria that areDivision of Biology,California Institute of these unusual behaviours, and do they have conse- small spheres or short rods — these are often referredTechnology, Pasadena, quences for mitochondrial function and cell physio­ to as ‘fragmented mitochondria’. In vivo, such changesCalifornia 91125, USA. logy? In this Review, we discuss the dynamic nature in dynamics control mitochondrial morphology dur-Correspondence to D.C.C. of mitochondria and summarize the mechanisms that ing development. For example, during Drosophilae-mail: dchan@caltech.edudoi:10.1038/nrm2275 drive mitochondrial fusion and fission. In addition, we melanogaster spermatogenesis, many mitochondriaPublished online discuss recent insights into how these processes affect synchronously fuse to form the Nebenkern structure,10 October 2007 the function of mitochondria. As well as controlling the which is required for sperm motility9.

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Box 1 | What happens to mitochondrial components after fusion? lines of evidence from neuronal studies suggest that mito- chondrial transport is regulated. First, mitochondria are recruited to regions with high energy demands, including active growth cones, presynaptic sites and postsynaptic + Wild type sites13,16,17. Such recruitment is regulated by neuronal acti- vation, further arguing that the recruitment of mitochon- dria is responsive to the local metabolic state. Second, neuronal mitochondria pause most often at sites that lack other mitochondria, resulting in a well-spaced axonal mitochondrial distribution14. Third, studies with the membrane-potential indicator dye JC‑1 suggest that mito- + Fusion mutant chondria with high membrane potential preferentially migrate in the anterograde direction, whereas mitochon- dria with low membrane potential move in the retrograde direction14. These migration patterns suggest that active In time-lapse movies of labelled mitochondria in living cells, mitochondria are observed to undergo cycles of fusion and fission. With each fusion event, two mitochondria are mitochondria are recruited to distal regions with high Nature Reviews | Molecular Cell Biology energy requirements, whereas impaired mitochondria merged into one. Intuitively, true fusion would be expected to involve outer membrane and inner membrane fusion, which would also result in mixing of the matrix contents. are returned to the cell soma, perhaps for destruction Indeed, these expectations have been experimentally confirmed. Apparent fusion events or repair. Finally, mitochondrial transport along axons that have been visualized in cells can be confirmed by a cell-hybrid mitochondrial fusion responds to local concentrations of nerve growth factor assay6,32 (see figure). In this assay, mitochondria in two distinct cell lines are differentially (NGF), suggesting that specific signalling pathways labelled with mitochondrially targeted green fluorescent protein (GFP) and DsRed. control mitochondrial recruitment and retention16,18. The cell lines are co-plated onto cover slips and exposed briefly to polyethylene glycol, a chemical that induces adjacent cells to fuse into cell hybrids. After a recovery period, Dynamic internal structure. In addition to changes in the cell hybrids are examined for mitochondrial fusion. In cell hybrids from normal cells, the overall shape of mitochondria, the internal structures mitochondrial fusion results in mitochondria that carry both GFP and DsRed (see figure, top). Cells that are defective for mitochondrial fusion form cell hybrids with distinct red of mitochondria are also dynamic. Three-dimensional and green mitochondria (see figure, bottom). A conceptually similar assay can be tomography of cryopreserved samples has provided new performed with yeast cells by allowing labelled yeast strains to mate and form zygotes4. views of inner membrane organization and plasticity19. By using matrix-targeted fluorophores, these assays show that mitochondrial fusion The inner membrane can be divided into distinct regions: results in mixing of the matrix contents. Moreover, by using mitochondrial markers that the inner boundary membrane, the cristae membrane are localized to the outer or inner membranes, fusion of the individual membranes can and the cristae junctions (FIG. 1c). The inner boundary be experimentally demonstrated; under normal conditions, outer and inner membrane membrane comprises the regions in which the inner fusion appear to be closely synchronized. membrane is in close proximity to the outer membrane. An important question is what happens to mitochondrial DNA (mtDNA) after fusion. These regions are probably important for protein import Each mitochondrion contains multiple copies of the mtDNA genome that are organized and might be the sites of coupled outer and inner mem- into one or more nucleoids. After fusion, these nucleoids appear to be motile and can potentially interact with each other73. In mammalian cells, mtDNA recombination has brane fusion. The cristae junctions are narrow ‘neck’ been documented, but its extent and importance is unclear. regions that separate the inner boundary membrane from the involuted cristae membrane. Cytochrome c, an intermembrane-space protein, is enriched in the space Dynamic subcellular distribution. Mitochondrial trans- that is encased by cristae membranes, and the regulated port is required to distribute mitochondria throughout opening of cristae junctions might be important for its the cell (FIG. 1b). In most cells, mitochondria are highly relocalization during apoptosis20. motile and travel along cytoskeletal tracks. Mitochondrial These regions of the mitochondrial inner membrane transport depends on the actin cytoskeleton in budding are not only morphologically distinct but also appear to yeast10 and on both actin and microtubules in mam- constitute separate functional domains. Proteins that malian cells3,11,12. Depending on the cellular context, are involved in the translocation of proteins through these transport processes can ensure proper inheritance the inner membrane, such as the TIM23 complex, are of mitochondria or can recruit mitochondria to active enriched in the inner boundary membrane, whereas regions of the cell. For example, in budding yeast, proteins that are involved in oxidative phosphorylationAnterograde mitochondria are transported into and retained in the are enriched in the cristae membranes21–23. In addition,The direction from the cell developing bud to ensure mitochondrial inheritance to the structure of mitochondrial membranes is linked to thebody towards the periphery. the daughter cell10. metabolic state of mitochondria (FIG. 1c). Purified mito- This regulation of mitochondrial distribution is par- chondria placed in low ADP conditions have limitedRetrograde ticularly evident in neurons. Quantitative measurements respiration and have an ‘orthodox’ morphology, charac-The direction from peripheralregions towards the cell body. of neuronal mitochondrial transport have reported rates terized by narrow cristae and few cristae junctions per ranging from 0.4 µm min–1 (Ref. 13) to 0.1–1 µm sec–1 cristae compartment. Under high ADP and substrateOxidative phosphorylation (refs 11,14,15). Such directed movements are not continu- conditions, isolated mitochondria have high respiratoryA biochemical pathway for ATP ous; rather, they are saltatory, with pauses often followed activity and a ‘condensed’ morphology, characterizedproduction that results inoxygen consumption and is by a reversal of direction. These patterns might reflect by larger cristae and several cristae junctions per cristaelocalized to the mitochondrial the attachment and detachment of cytoskeletal motors. compartment19. It is unknown how inner membranescristae. Although these movements can appear chaotic, several convert between these states, but inner membrane fusion

a member of the mitofusin family of GTPases. The yeast

Fusion orthologue, Fzo1, has a conserved role in mitochondrial + fusion24, and genetic screens in yeast have identified Fission additional modulators of mitochondrial fusion and fission2,25 (FIG. 3b). The core machineries that mediateb mitochondrial fusion and fission are best understood Anterograde in yeast. Several of these components have functionally movement conserved mammalian homologues. More compre- hensive discussions of the molecular mechanisms of Nucleus mitochondrial fusion and fission have been presented in recent reviews (for example, see REF. 1).

Retrograde Mitochondrial fusion. In yeast, the core mitochondrial

movement fusion machinery consists of two GTPases: Fzo1 andc Mgm1 (FIG. 3). Fzo1 is located on the mitochondrial outer IMS OM membrane and is essential for fusion of the outer mem- branes24,26. The mammalian homologues of Fzo1 are the CJ IBM IM mitofusins MFN1 and MFN2. These two related proteins form homo-oligomeric and hetero-oligomeric complexes CM that are essential for fusion6,27,28. Mitofusins are required on adjacent mitochondria during the fusion process, ‘Condensed’ morphology ‘Orthodox’ morphology implying that they form complexes in trans between apposing mitochondria26,29. A heptad repeat region of MFN1 has been shown to form an antiparallel coiled coil that is probably involved in tethering mitochondria

Low [ADP] during fusion29. Mgm1 is a dynamin-related protein that is essential for fusion of the mitochondrial inner membranes in High [ADP] yeast30, a function that is consistent with its localization to the intermembrane space and its association with the inner membrane. The mammalian orthologue OPA1 is also essential for mitochondrial fusion28,31. In yeast,Figure 1 | Mitochondria as dynamic organelles. a | Mitochondrial fusion and fission the outer membrane protein Ugo1 physically links Fzo1control mitochondrial number and size. With fusion, Nature two mitochondria becomeCell a single and Mgm1, but no mammalian orthologue has yet been Reviews | Molecular Biologylarger mitochondrion with continuous outer and inner membranes. Conversely, a single discovered2.mitochondrion can divide into two distinct mitochondria by fission. b | In mammalian The membrane potential across the mitochondrialsystems, mitochondria are distributed throughout the cytoplasm by active transport along inner membrane is maintained by the electron trans-microtubules and actin filaments. Distinct molecular motors transport the mitochondria in port chain and is essential for mitochondrial fusion26,32.anterograde or retrograde directions. c | Inner membrane dynamics. The diagram Ionophores that dissipate the mitochondrial membraneindicates the different regions of the inner membrane. The bottom panels show electron potential cause mitochondrial fragmentation, owing tomicroscopy (EM) tomograms of two mitochondria under different metabolic conditions(red, outer membrane; yellow, inner boundary membrane; green, cristae membrane). an inhibition of mitochondrial fusion32,33. In an in vitroCristae organization can vary widely, often in response to the bioenergetic state of the fusion assay, both the proton and the electrical gradientcell: an ‘orthodox’ cristae morphology, with narrow cristae and few cristae junctions per components of the membrane potential are important26.cristae compartment, is found in low ADP conditions, whereas a ‘condensed’ morphology, The mechanistic link between membrane potential andwith larger cristae and several junctions per cristae compartment, is found in high fusion remains to be resolved, but one factor appears toADP conditions19. EM images reproduced with permission from Ref. 19  (2006) Elsevier. be the dependence of post-translational processing ofCJ, cristae junction; CM, cristae membrane; IBM, inner boundary membrane; IM, inner OPA1 on the membrane potential34.membrane; IMS, intermembrane space; OM, outer membrane. Recent work has also identified mitochondrial lipids as important factors in fusion. Mitochondrial morphol- ogy screens in yeast identified members of the ergosterol and fission might be involved19. Taken together, these synthesis pathway as being required for normal mito- observations indicate that inner membrane morphology chondrial morphology35,36. Recently, mitochondrial is intimately related to bioenergetics, although the causal phospholipase D has been identified as a protein that isCoiled coil relationship remains unclear. important for mitochondrial fusion37. This mitochon-A structural motif that isformed by polypeptide drial outer membrane enzyme hydrolyses cardiolipin tosequences that contain Mediators of fusion and fission generate phosphatidic acid. Interestingly, ergosterol hashydrophobic heptad repeats. Molecular analysis of mitochondrial morphology began been associated with yeast vacuole fusion38, and phos- with the discovery in 1997 of the D. melanogaster fusion phatidic acid is thought to play a part in generating theDynaminA large GTPase that is thought factor fuzzy onions (FZO), a mitochondrial outer mem- membrane curvature that is required for SNARE-mediatedto mediate vesicle scission brane GTPase that is required for the fusion of mito- fusion39. Thus, specific lipids might have similar roles induring endocytosis. chondria during spermatogenesis9. FZO is the founding distinct types of membrane fusion.

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No fusion Wild type No fission This modification of DRP1 might affect its association Mfn-null DRP1 K38A with mitochondrial membranes. Mitochondrial fission is also regulated by the cell cycle. For example, mitochondria in HeLa cells are usually tubular, but they become more fragmented during mitosis, a phenomenon that might facilitate the partitioning of mitochondria to daughter cells during cytokinesis. This regulated fragmentation of mitochondria is due to increased mitochondrial fission, and phosphorylation of DRP1 during mitosis has been implicated55.Figure 2 | Mitochondrial fusion and fission regulate morphology. Mitochondrial In addition to the genes that encode core fusion andlength, size and connectivity are determined by theNature relativeReviews rates of| Molecular Cell Biology mitochondrial fusion fission components, other genes can affect mitochon-and fission. In wild-type cells (shown in the central panel), mitochondria form tubules of drial morphology. Large-scale visual screens for aberrantvariable length. In the absence of mitochondrial fusion (for example, in mitofusin (Mfn)- mitochondrial morphology in mutant yeast have yieldednull cells (shown in the left panel), which lack MFN1 and MFN2), unopposed fission resultsin a population of mitochondria that are all fragmented. Conversely, decreased fission numerous genes of interest and provided general insightsrelative to fusion (for example, in DRP1 K38A cells (shown in the right panel), which have into the control of mitochondrial morphology35,36. Thesea dominant-negative form of dynamin-related protein-1 (DRP1)) results in elongated and screens suggest that several cellular pathways influencehighly interconnected mitochondria. Scale bar represents 10 µm. mitochondrial morphology and inheritance, including ergosterol biosynthesis, mitochondrial protein import, actin dynamics, vesicular fusion and ubiquitin-mediatedMitochondrial membrane Mitochondrial fission. Mitochondrial fission requires protein degradation. The close interplay between mito-potential the recruitment of a dynamin-related protein (Dnm1 in chondrial protein import and morphology has beenThe electrochemical gradient yeast and DRP1 in mammals) from the cytosol (FIG. 4). emphasized by the recent finding that the mitochondrialthat exists across the Both Dnm1 and DRP1 assemble into punctate spots on morphology genes MMM1, MDM10 and MDM12 have amitochondrial innermembrane. mitochondrial tubules, and a subset of these complexes direct role in the assembly of β‑barrel proteins in the outer lead to productive fission events5,7,8. By analogy with the mitochondrial membrane56.Ergosterol classical function of dynamin in endocytosis, Dnm1A steroid compound that is a and DRP1 are thought to assemble into rings and spirals Proteins that are required for mitochondrial transport.component of yeast cell that encircle and constrict the mitochondrial tubule Energy-dependent molecular motors transport mitochon-membranes and which mighthave a role similar to that of during fission25. Consistent with this model, purified dria along cytoskeletal filaments. Along micro­tubules,cholesterol in mammalian cell Dnm1 can indeed form helical rings and spirals in vitro, multiple kinesin family members and cytoplasmic dyneinmembranes. with dimensions that are similar to those of constricted have been implicated in anterograde and retrograde mitochondria40. Moreover, Dnm1 assembly is required mitochondrial transport, respectively3. Recent work hasSNARE for fission activity41. clarified the linkage between mitochondria and kinesin‑1.(soluble N‑ethylmaleimide-sensitive fusion protein (NSF) The recruitment of Dnm1 to yeast mitochondrial fis- Genetic screens in D. melanogaster identified miltonattachment protein (SNAP) sion sites involves three other components. One of these and Miro, both of which are required for anterogradereceptor). A highly α‑helical is Fis1, a mitochondrial integral outer membrane protein mitochondrial transport in neurons57,58. Milton interactsprotein that mediates the that is essential for fission42–44. Fis1 binds indirectly to directly with kinesin and Miro, which is a mitochondrialspecific fusion of vesicles with Dnm1 through one of two molecular adaptors, Mdv1 outer membrane protein that has GTPase and Ca2+-bind-target membranes. or Caf4 (Ref. 45) (FIG. 4b). Either Mdv1 or Caf4 is suffi- ing EF‑hand domains59. In yeast, disruption of the MiroF-box protein cient to allow the Fis1-dependent recruitment of Dnm1, orthologue Gem1 results in abnormalities in mitochon-A protein containing an F-box although Mdv1 has a more important role in mediating drial morphology and poor respiratory activity60. Bothmotif, a small domain that is fission. FIS1, the mammalian homologue of Fis1, is also GTP-binding and Ca2+-binding motifs are essential forused for protein interactions. essential for mitochondrial fission46, but no homologues Gem1 function, which appears not to be involved inThe best-characterized F-boxproteins are components of an of Mdv1 or Caf4 are currently known. FIS1 and DRP1 fusion or fission. Depending on the cell type, mitochon-E3 ubiquitin ligase, and help in are also required for the fission of peroxisomes47,48. dria can also travel along actin filaments under the controlubiquitin-dependent protein of myosin motors3.degradation by recognizing Other regulators of dynamicsspecific substrates. Mitochondrial fusion and fission activities are probably Proteins that mediate inner membrane morphology.β-barrel protein coordinated with cellular physiology. In yeast, the steady- Studies of mitochondrial inner membrane structure areA protein composed of a state levels of Fzo1 are controlled by the F‑box protein complicated by the intimate link between mitochondrialβ-sheet that is rolled up Mdm30, which negatively regulates Fzo1 levels in a bioenergetics and cristae structure. As a result, disruptioninto a cylinder. One such proteasome-independent manner49,50. In mammalian of the proteins that are important for bioenergetics canmitochondrial β-barrel proteinis VDAC (voltage-dependent cells, post-translational modification of DRP1 regulates its lead to a secondary effect on inner membrane structure.anion channel), which forms a function in mitochondrial fission. The mitochondrial E3 Nevertheless, several proteins probably have a specificpore in the outer membrane. ubiquitin ligase MARCH5 is essential for mitochondrial role in controlling cristae structure. In addition to their fission51. This requirement is probably related to the abil- roles in mitochondrial fusion, Mgm1 and OPA1 areKinesin ity of MARCH5 to promote DRP1 ubiquitylation and to important for cristae structure. Loss of Mgm1 in yeastA microtubule-based molecularmotor protein that is most associate physically with ubiquitylated DRP1 (Refs 52,53). or knockdown of OPA1 in mammalian cells results inoften directed towards the plus Furthermore, during apoptosis, sumoylation of DRP1 is disorganized inner membrane structures30,61–64. In bothend of microtubules. activated in a BAX- and/or BAK-dependent manner54. cases, homo-oligomeric interactions are involved30,64.

Biological functions of mitochondrial dynamics

Why do mitochondria continually fuse and divide? Ugo1 Recent studies show that these processes have impor- tant consequences for the morphology, function and s-Mgm1 distribution of mitochondria. First, fusion and fission OM I-Mgm1 control the shape, length and number of mitochondria. IMS The balance between these opposing processes regulates mitochondrial morphology. Second, fusion and fission allow mitochondria to exchange lipid membranes and IM intramitochondrial content. Such exchange is crucial for maintaining the health of a mitochondrial population. Third, the shape of mitochondria affects the ability of Cristae cells to distribute their mitochondria to specific sub­ cellular locations. This function is especially important Figure 3 | Mitochondrial fusion. a | Mitochondrial fusion in highly polarized cells, such as neurons. Finally, mito- consists of outer membrane (OM) fusion followed by inner chondrial fission facilitates apoptosis by regulating the membrane (IM) fusion. Normally Nature these Reviews events occur | Molecular Cell Biology release of intermembrane-space proteins into the cytosol. coordinately. b | The dynamin-related proteins Fzo1 and As a result of these cellular functions, mitochondrial Mgm1 are key molecules in the yeast mitochondrial fusion dynamics has consequences for development, disease machinery. Fzo1 is an integral outer membrane protein and apoptosis. with GTPase and heptad repeat domains that face the cytoplasm. All of the domains are required for the fusion Maintaining a healthy mitochondrial population. activity of Fzo1. Mgm1 is present on the inner membrane, Mitochondrial fusion is required to maintain a func- facing the intermembrane space (IMS), and is proteolytically processed by a rhomboid protease. Both tional mitochondrial population in the cell. Fibroblasts long (l-Mgm1) and short (s-Mgm1) forms are required for that lack both MFN1 and MFN2 have reduced respira- mitochondrial fusion. In addition to inner membrane tory capacity, and individual mitochondria show great fusion, Mgm1 is required for the maintenance of cristae heterogeneity in shape and membrane potential28. Cells structures. Ugo1 binds to both Fzo1 and Mgm1 and that lack OPA1 show similar defects, with an even greaterDynein probably coordinates their function. All components are reduction in respiratory capacity.A microtubule-based molecular encoded by nuclear DNA. The mitofusin proteins MFN1 How does fusion protect mitochondrial function?motor that is directed towards and MFN2 are the mammalian homologues of Fzo1; OPA1 It is probable that a primary function of mitochondrialthe minus end of microtubules. is the mammalian homologue of Mgm1. No mammalian fusion is to enable the exchange of contents betweenEF-hand domain homologue of Ugo1 has been identified so far. mitochondria (BOX 1). As a result, mitochondria shouldA helix-loop-helix protein motifthat can bind a Ca2+ ion. not be considered autonomous organelles; instead, the hundreds of mitochondria in a typical cell exist asMitochondrial F1F0 ATP Mitochondrial F 1FoATP synthase , a rotary enzyme a population of organelles that cooperate with eachsynthase embedded in the inner membrane that couples proton other through fusion and fission. The heterogeneousA large, multisubunit enzyme pumping to ATP synthesis, is essential for normal cristae properties of mitochondria in fusion-deficient cellsembedded in themitochondrial cristae that uses structure65. This role in inner membrane structure are consistent with this model28. In normal cells, a fewthe proton gradient across the involves a dimeric form of ATP synthase that contains mitochondria might be non-functional owing to the lossinner membrane to synthesize the additional subunits e and g. As visualized by elec- of essential components. However, this dysfunction isATP. tron microscopy, the ATP synthase dimer has a dimeric transient because mitochondrial fusion provides a path-Mitochondrial DNA interface with a sharp angle that could distort the local way for these defective mitochondria to regain essential(mtDNA). A circular genome lipid membrane. This distortion might contribute to components (FIG. 5a).(~16 kb in mammals) located the high membrane curvature that characterizes cristae An essential component of mitochondrial function isin the mitochondrial matrix tubules66,67. Mgm1 is required for the oligomerization mitochondrial DNA (mtDNA), which is organized intothat encodes 13 polypeptides of ATP synthase, providing a link between these two compact particles termed nucleoids. The mtDNA genomeof the electron transport chain,22 tRNAs and 2 rRNAs. modulators of cristae structure63. encodes essential subunits of the respiratory complexes Additional proteins modulate inner membrane I, III and IV, and is therefore essential for oxidative phos-Nucleoid dynamics. In yeast, Mdm33 is required for normal phorylation. When mitochondrial fusion is abolished,A compacted mass of DNA. mitochondrial morphology and its overexpression leads a large fraction of the mitochondrial population losesMitochondrial DNA isorganized into nucleoids, each to septation and vesiculation of the inner membranes68. mtDNA nucleoids72. During mitochondrial division inconsisting of several Because of these phenotypes, Mdm33 has been suggested normal cells, most daughter mitochondria inherit atmitochondrial genomes. to have a role in inner membrane fission. Knockdown of least one mtDNA nucleoid73. However, in cases where a

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Mitochondrial distribution and recruitment in neurons.

a Dnm1 b Given the importance of mitochondrial dynamics in Dnm1 maintaining bioenergetics, these dynamics are probably a ubiquitous phenomenon that is important for all cells. However, certain cells, particularly neurons, seem to be especially dependent on its proper control. This depend- Adaptor poteins (Mdv1, Caf4) ence of neurons probably stems from their high energy demands and the special importance of proper mito- chondrial distribution: mitochondria are concentrated in Fis1 several neuronal regions, including pre- and postsynaptic sites13,17. To achieve this non-uniform distribution, neurons OM rely heavily on active transport to recruit mitochondria IM and other organelles to nerve terminals3.Figure 4 | Mitochondrial fission. a | In yeast, mitochondrial fission is mediated by the The proper localization of mitochondria to axondynamin-related protein Dnm1. Cytoplasmic Dnm1Nature Reviews localizes Molecular Cell outer to the| mitochondrial Biology terminals depends on mitochondrial dynamics. Neuronsmembrane (OM), where it oligomerizes into a ring structure that constricts and severs the that lack Milton, Miro or DRP1 show defective mito-mitochondrion. In this model, Dnm1 functions in an analogous manner to the way chondrial transport and have sparse mitochondria atdynamin functions in endocytosis. b | The localization of Dnm1 on the mitochondrial outer axon terminals. Such distribution defects lead to reducedmembrane is mediated by Fis1 and the adaptor proteins Mdv1 and Caf4. Fis1 is an integral capacity for synaptic transmission57,58,78. It seems likelyouter membrane protein that interacts with the N termini of Mdv1 and Caf4. Both Mdv1and Caf4 have C‑terminal WD‑40 repeats that bind Dnm1. Fis1 and Dnm1 have that mitochondria that are localized to synapses aremammalian homologues (FIS1 and DRP1, respectively), but no Mdv1 or Caf4 homologues primarily required to drive ATP-dependent processes.have been identified so far. IM, inner membrane. The synapses of neurons that express mutant DRP1 show defective mobilization of the reserve vesicle pool (an ATP- dependent process), and the defects in synaptic transmis- daughter fails to inherit a nucleoid, mitochondrial fusion sion can be rescued by experimentally filling synapses would enable restoration of mtDNA. In fusion-deficient with ATP. In addition, synapse-localized mitochondria cells, the lack of content exchange prevents restoration help to regulate Ca2+ homeostasis, although this function of mtDNA nucleoids and probably accounts for the appears to be crucial only during intense synaptic activity. heterogeneity in membrane potential and the reduced Synapses that lack Miro or DRP1 have elevated resting respiratory capacity. It should be noted that fusion- Ca2+ levels, but normal Ca2+ dynamics are maintained deficient cells still maintain significant numbers of except under sustained nerve stimulation57,78. mtDNA nucleoids; however, due to ongoing mitochon- Both mitochondrial fusion and fission affect the drial fission, these nucleoids are encased by a small mitochondrial distribution in dendrites. In hippocampal mitochondrial mass, and therefore the functional mito- neurons, mitochondria accumulate at dendritic spines chondrial mass (at least in terms of bioenergetics) in such following neuronal stimulation13. Inhibition of mito- cells is greatly reduced (FIG. 5b). In addition to mtDNA, chondrial fission causes elongation of the mitochondria it is also possible that other components, such as sub- and decreases the abundance of dendritic mitochondria and strates, metabolites or specific lipids, can be restored in the density of dendritic spines. Conversely, increased defective mitochondria by fusion. Further studies will fission facilitates the mobilization of dendritic mitochon- determine whether content exchange is the primary dria and leads to an increased spine number13. In the function of mitochondrial fusion. The importance of cerebellum, the distribution of mitochondria in the den- mitochondrial fusion in development and disease might dritic processes of Purkinje neurons is highly dependent be a consequence of this function. on mitochondrial fusion72 (see below).

mitochondrial dynamics result in specific developmental appears to be important for proper mitochondrial defects. Mice that lack either MFN1, MFN2 or OPA1 fail redistribution in lymphocytes during chemotaxis 79. to survive past mid-gestation6,74,75. MFN2 has a highly Mitochondria are concentrated in the trailing edge in specific function in the development of the trophoblast lymphocyte cell lines that migrate in response to chemical giant cell layer of the placenta6. Likewise, MFN1 appears attractants. Modulation of mitochondrial fusion or to have an essential placental function72. fission affects both mitochondrial redistribution and Mitochondrial fission is also an essential process. cell migration. Fragmentation enhances mitochondrial Worms that are deficient in mitochondrial division die redistribution and cell migration, whereas conditions that before adulthood76. An infant patient with a dominant- promote fusion have the opposite effect. Therefore, as in negative DRP1 allele has been reported. This patient died neurons, mitochondrial shape in lymphocytes can affect at ~1 month of age and had a wide range of abnorm­ the recruitment of mitochondria to local cellular areas. alities, including reduced head growth, increased lactic acid and optic atrophy. Fibroblasts from this patient Regulation of apoptosis. In apoptosis, several structuralChemotaxisThe directed movement of cells showed elongated mitochondria and peroxisomes 77. changes occur in mitochondria during the early phase ofin response to a chemical It is unclear how the developmental defects are related cell death (FIG. 6). The mitochondria become fragmentedstimulus. to these organellar shape changes. owing to increased fission activity. At approximately the

a Wild-type cells b Fusion-deficient cells can occur in the absence of mitochondrial fission. An important issue to resolve in future studies is how fission is related to the permeabilization of mitochondria. Surprisingly, the apoptotic proteins BAX and BAK, Fusion which have well-established pro-apoptotic roles in mitochondrial membrane permeabilization, also appear to regulate mitochondrial morphology. BAX and BAK Defective double-knockout cells have fragmented mitochondria mitochondrion due to reduced mitochondrial fusion87, although the extent of this effect depends on the experimental sys- tem88. Little is known about how BAX and BAK mediate their effects on mitochondrial morphology, but BAX influences MFN2 distribution on the mitochondrial outer membrane87 and BAK associates with MFN1 and Fission MFN2 (Ref. 88). In conjunction with MOMP, remodelling of the cristae membranes is required for the rapid and efficient release of cytochrome c20,89. Most cytochrome c is localized to cristae compartments20; OPA1 appears to regulate the Rescued diameter of cristae junctions and therefore regulates mitochondrion cytochrome c release 64,90. Overexpression of OPA1Figure 5 | Mitochondrial dynamics protects mitochondrial function. a | In wild-type blocks cytochrome c release following the induction Nature Reviews | Molecular Cell Biology of apoptosis by maintaining narrow cristae junctions64.cells, the vast majority of mitochondria are functional (shown in green). In this simplifieddiagram, one mitochondrion is depicted as non-functional (shown in orange). One of DRP1 has also been proposed to play a part in cristaeseveral possible reasons for dysfunction is a lack of mitochondrial DNA (mtDNA) remodelling during apoptosis91.nucleoids (shown as black circles). The dysfunctional mitochondrion can regain itsfunction and mtDNA by fusing with a neighbouring mitochondrion. The fused Role in human diseasemitochondrion then undergoes fission, with both daughter mitochondria receiving Several human diseases are caused by mutations in genesmtDNA nucleoids. It should be noted that the identities of the daughter mitochondria that are essential for mitochondrial dynamics (TABLE 1).are distinct from the parental mitochondria, owing to content exchange and the fact Each of these diseases causes degeneration of specificthat the fission point is typically distinct from the fusion point. b | In fusion-deficientcells, mitochondria are fragmented due to ongoing fission in the absence of fusion. nerves, reinforcing the notion that neurons are particularlyMitochondria that lack mtDNA nucleoids accumulate because there is no pathway for prone to defects in mitochondrial dynamics.defective mitochondria to regain mtDNA. Fusion-deficient cells can maintain mtDNAnucleoids, but such nucleoids serve a much smaller mitochondrial mass. OPA1 and autosomal dominant optic atrophy. Heterozygous mutations in OPA1 cause autosomal dominant optic atrophy (ADOA), the most common same time, mitochondrial outer membrane permeabilization heritable form of optic neuropathy92,93. This disease is (MOMP) causes the release of contents of the inter- characterized by the degeneration of retinal ganglion membrane space, such as cytochrome c and second cells, the axons of which form the optic nerve. More than mitochondria-derived activator of caspase (SMAC)/ 100 pathogenic OPA1 mutations have been reported, Diablo, into the cytoplasm. Because cytochrome c is with most occurring in the GTPase domain94. Half of the preferentially sequestered in cristae compartments, mutants encode a truncated protein owing to a nonsense it is thought that the opening of cristae junctions is mutation. A few nonsense mutations abolish nearly the a vital step in facilitating its efficient release. Once in entire coding sequence, suggesting that haploinsufficiency the cytosol, cytochrome c activates a cascade of caspases of OPA1 can cause ADOA. It remains possible that other, that propagate and execute the apoptotic programme. less severe, truncations might have dominant-negative These three structural changes — fragmentation, activity. MOMP and cristae remodelling — occur at similar How these OPA1 mutations cause the clinical symp- times, but their temporal sequence and causative links toms of ADOA remains to be clarified. Non-neuronalMitochondrial outermembrane permeabilization are still controversial80,81. cells from patients with ADOA can have aggregated, frag-(MOMP). The opening of pores Mitochondrial fragmentation during apoptosis is mented or normal mitochondria93,95; however, becausein the mitochondrial outer associated with dynamic changes in the mitochondrial data from only a few patients have been reported, it is notmembrane — an early event localization of several proteins, including BAX, BAK, clear whether these findings are the norm. In addition,during apoptosis that releases MFN2, endophilin and DRP1 (Ref. 81). Inhibition of OPA1 mutations have been associated with reduced ATPapoptotic factors from themitochondrial intermembrane fission activity blocks mitochondrial fragmentation, production and reduced mtDNA content96,97. The defectsspace. reduces cytochrome c release and can reduce or delay that have been documented in human ADOA diseased cell death depending on the experimental system46,82,83. tissue are not as severe as those observed in experimentalHaploinsufficiency In Caenorhabditis elegans and D. melanogaster, disrup- cells in which OPA1 is depleted. Fibroblasts that are defi-A genetic state in diploids inwhich a single functional copy tion of DRP1 reduces the number of cell deaths84–86. cient for OPA1 have fragmented mitochondria, defectsof a gene is insufficient to In multiple systems, it seems that fission is important in respiration, aberrant cristae structure and increasedmaintain a normal phenotype. for rapid and efficient cell death, although apoptosis susceptibility to apoptosis28,31,62,98.

876 | november 2007 | volume 8 www.nature.com/reviews/molcellbio

Cytochrome c occur in each of the heptad repeat domains of MFN2. In

addition to the loss of peripheral nerve function, a subset of patients with CMT2A have optic atrophy, suggesting that OPA1 and MFN2 mutations can lead to overlapping Apoptotic clinical outcomes101,102. stimuli Because of the difficulties in studying nerve tissue from patients, the pathogenic mechanisms that lead to peripheral nerve degeneration in CMT2A are not well understood. Only one study has reported ultrastructural Cristae junctions defects in mitochondria from the nerves of patients with CMT2A. Mitochondria in the sural nerve of two patients showed structural aberrations in their outer and innerFigure 6 | Mitochondrial dynamics during apoptosis. At an early stage of apoptosis, membranes, along with swelling that is suggestive of Nature Reviewsthree structural changes occur in mitochondria. Fragmentation | Molecular takes place asCell Biology a result of mitochondrial dysfunction103. Aggregation of mitochon-increased fission mediated by dynamin-related protein-1 (DRP1) and the mitochondrial dria was also observed. Interestingly, CMT2A alleles offission-1 protein (FIS1). Mitochondrial outer membrane permeabilization (MOMP; MFN2 (Refs 27,104) cause mitochondrial aggregationindicated by dashed outlines) is induced by the pro-apoptotic BCL2-family members BAX and subsequent mitochondrial transport defects inand BAK. MOMP enables the release of cytochrome c (shown as red dots) and other soluble neurons104. However, the mitochondrial aggregationproteins from the intermembrane space. However, release of cytochrome c is efficient only phenotype is dependent on significant overexpression27,if the cristae junctions are widened to allow escape from the cristae compartments. The and therefore its relevance to disease pathogenesisdynamin-related proteins OPA1 and DRP1 have been implicated in cristae remodelling. remains to be clarified. Several perplexing issues remain to be resolved con- cerning the molecular genetics of CMT2A. How does Mouse models of ADOA that contain OPA1 muta- mutation of one copy of MFN2 lead to disease? Why are tions develop the features of ADOA in an age-dependent long peripheral neurons selectively affected, given that manner74,75. Heterozygous mice show a progressive MFN2 is a broadly expressed protein? Clues to these decline in retinal ganglion cell number and aberrations issues have come from analysis of CMT2A alleles in of axons in the optic nerve. Mice that are homozygous for mice27. Many CMT2A alleles of Mfn2 are non-functional the OPA1 mutation die at mid-gestation74,75, which is con- for fusion when expressed alone. However, the fusion sistent with an essential requirement for mitochondrial activity of these non-functional alleles can be efficiently fusion during embryonic development6. complemented by wild-type MFN1 (but not MFN2). This complementation is due to the ability of MFN1 and MFN2 and Charcot-Marie-Tooth 2A. Charcot-Marie- MFN2 to form hetero-oligomeric complexes that are Tooth (CMT) disease, one of the most common heredi- functional for fusion. In a patient with CMT2A, there- tary neuropathies, is caused by mutations in at least 30 fore, cells that express MFN1 are protected from gross different genes99. Affected individuals have progressive loss of fusion activity. By contrast, cells with little or no distal motor and sensory impairments that start in MFN1 expression suffer a greater relative loss of fusion the feet and hands as a result of the degeneration of the activity. In part, these properties of the CMT2A alleles long peripheral nerves. Depending on the type of CMT, might underlie the selective loss of sensory and motor these diseases are caused by either a primary defect in neurons. Consistent with this model, MFN2 seems to be the Schwann cells that myelinate the peripheral nerves more highly expressed in central and peripheral nervous or by a defect in the neurons themselves99. CMT2A is tissue than MFN1 (S.A.D. and D.C.C., unpublished obser-Sural nerve an axonopathy that is caused by the latter type of defect, vations). Even in the peripheral nerves, it appears thatA sensory nerve innervating and it has been associated with >40 mutations in MFN2. mitochondrial fusion defects are only partial because onlythe calf and foot that iscommonly investigated by Nearly all of these disease alleles contain missense muta- the longest nerves are affected. Most probably, the extremebiopsy for the evaluation of tions or short, in-frame deletions100. Most mutations dimensions of the long peripheral nerves make them mostperipheral neuropathies. cluster in or near the GTPase domain, but some also vulnerable to changes in mitochondrial dynamics. How might perturbations in mitochondrial fusion lead to neurodegeneration? Clues to the pathogenic Table 1 | Disorders associated with mitochondrial perturbations mechanisms have come from the finding that mice Disease Mitochondrial Gene Description that lack MFN2 show highly specific degeneration of function Purkinje neurons in the cerebellum, resulting in cere­ CMT2A Fusion MFN2 Autosomal dominant bellar ataxia72. Purkinje cells are the sole efferent neurons peripheral neuropathy of the cerebellum, and they have exquisitely formed den- ADOA Fusion OPA1 Autosomal dominant optic dritic processes. Both developing and mature Purkinje atrophy (ADOA) cells that lose MFN2 fail to support dendritic outgrowth, CMT4A Fission? GDAP1 Autosomal recessive particularly that of dendritic spines, which are the sites of peripheral neuropathy synaptic connections. In normal Purkinje cells, abundant Unnamed Fission DRP1 Neonatal lethality tubular mitochondria reside in dendritic processes. By CMT, Charcot-Marie-Tooth; DRP1, dynamin-related protein-1; GDAP1, ganglioside-induced contrast, mutant Purkinje cells have fragmented mito- differentiation-associated protein-1; MFN2, mitofusin-2; OPA1, optic atrophy-1. chondria that fail to distribute effectively along dendritic

processes. In addition, the Purkinje cells show a loss of Perspectives

respiratory activity, probably owing to an accumulation The study of mitochondrial dynamics has undergone of mitochondria that lack mtDNA nucleoids. Therefore, great advances in the past few years. It is now clear that loss of mitochondrial fusion in Purkinje neurons impairs mitochondrial dynamics is important for the functional respiratory activity and mitochondrial localization. state of mitochondria. By enabling content exchange between mitochondria, fusion and fission prevent the GDAP1 and Charcot-Marie-Tooth 4A. Another form of accumulation of defective mitochondria. These oppos- CMT is associated with defects in mitochondrial dyn­ ing processes also control mitochondrial shape, which amics. Ganglioside-induced differentiation-associated affects the distribution of mitochondria as well as their protein-1 (GDAP1) is mutated in CMT4A, one of the participation in apoptosis. As a result, mitochondrial few recessive forms of CMT disease. CMT4A has both dynamics is particularly important in cells and tissues demyelinating and axonal features and, consistent with that have a special dependence on mitochondrial func- this mixed clinical presentation, GDAP1 is expressed tion. Defects in mitochondrial dynamics can manifest in in both Schwann cells and neurons105. GDAP1 is an mammalian development, apoptosis and disease. As our integral outer membrane protein that probably affects knowledge of mitochondrial dynamics increases, we can mitochondrial division105. Disease alleles either fail to expect to learn about its involvement in other processes. localize to mitochondria or are defective in stimulating The link between defects in mitochondrial fusion and mitochondrial fission when overexpressed105. If GDAP1 neurodegenerative disease is particularly intriguing. In disease alleles disrupt normal mitochondrial fission, future studies, the pathophysiological mechanisms that they might cause mitochondrial distribution defects underlie neurodegenerative diseases such as ADOA and similar to those that are induced by the DRP1 mutations CMT2A will hopefully be further dissected in appropriate discussed above13,78. animal models.